Fibroblast-selective smoothened governs the prognosis of acute kidney injury

doi:10.21203/rs.3.rs-1999704/v1

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Fibroblast-selective smoothened governs the prognosis of acute kidney injury

https://doi.org/10.21203/rs.3.rs-1999704/v1

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The smoothened (Smo) receptor facilitates hedgehog signaling between kidney fibroblasts and tubules during acute kidney injury (AKI). Tubule-derived hedgehog is protective in AKI, but the role of fibroblast-derived Smo is unclear. Here, we report that Smo ablation in fibroblasts mitigated tubular cell apoptosis and inflammation, enhanced perivascular mesenchymal activities, and preserved kidney function after AKI. Global proteomics of these kidneys identified extracellular matrix proteins, and nidogen-1 glycoprotein in particular, as key response markers; Intriguingly, Smo was bound to nidogen-1 in cells, suggesting that loss of Smo could impact nidogen-1 accessibility. Phosphoproteomics revealed that the ‘AKI protector’ Wnt pathway was activated in these kidneys, and in vitro and ex vivo, nidogen-1 was able to induce Wnts and repress tubular cell apoptosis. Altogether, our results support that fibroblast-derived Smo dictates AKI fate through cell-matrix interactions, including nidogen-1, and establish a robust resource and path to further dissect AKI pathogenesis.

Acute kidney injury

smoothened

nidogen-1

wnt

Acute kidney injury (AKI) is a clinical syndrome characterized by an abrupt decline in renal function within a few hours or days, which is associated with high morbidity and mortality globally [1]. Although developing effective treatments for human AKI remains a challenge, advances in research have widened our knowledge of the pathogenesis of this refractory disease. In AKI induced by ischemia, sepsis, nephrotoxins, or major surgery, renal tubules have been the well-acknowledged epicenter of damage [2]. However, emerging evidence shows that resident perivascular mesenchymal cells, such as fibroblasts and pericytes, are instinctively mobilized to participate in the repair process once AKI occurs [3, 4]. Unfortunately, the role of fibroblasts in AKI remains a mystery.

Fibroblasts are becoming increasingly appreciated for their association with AKI prognosis [5–7]. Studies have demonstrated that after AKI, fibroblast activation precedes tubular cell proliferation [4]. Early activated fibroblasts immediately release signals to neighboring cells, such as tubular epithelial or other perivascular mesenchymal cells, to rescue kidneys from AKI through auto- or paracrine mechanisms. Several highly conserved cell signaling systems are recruited for the acute phase of kidney injury/repair, including Hedgehog (Hh), Wnt, Notch, and mammalian target of rapamycin [3, 8–14]. Moreover, fibroblasts are the principal effector cells responsible for synthesizing the extracellular matrix (ECM) [15]. After AKI, appropriate types and amounts of ECM are beneficial for forming a favorable microenvironment in the diseased kidneys [16]. This microenvironment functionally serves as a physical home to allow injured cells to rest and as a scaffold backbone for tissue remodeling. Therefore, it is conceivable to speculate that fibroblasts have a unique role in regulating AKI recovery.

Smoothened (Smo) is a seven-pass transmembrane receptor that is a crucial positive regulator in the Hh signaling pathway. The Hh pathway is a highly evolutionarily conserved system linked to cell-cell communications for embryonic development and adult tissue homeostasis [17, 18]. Its activity is triggered by stoichiometric binding of Hh ligands to Patched (Ptc) [19]. Ptc itself is an inhibitory transmembrane receptor that keeps the Hh pathway off by inhibiting Smo in the absence of Hh [20]. Once Hh is presented to Ptc, Smo is activated which turns on downstream genes such as the glioma-associated oncogene homolog (Gli) transcription factor. Mammals have three Hh homologues, Desert (Dhh), Indian (Ihh), and Sonic (Shh), of which Shh is the best studied [21, 22]. In the kidney, Shh is exclusively produced by tubules [4, 23], but fibroblasts are its specific target.[24] In these responsive fibroblasts, how Smo regulates AKI remains unclear. Besides transducing signals, we ask whether fibroblast-derived Smo directly impacts kidney protection by binding small molecules or large proteins to adjust cell behaviors and even the tissue microenvironment after AKI. Here, integrating traditional molecular pathology with systems biology, we illustrate how fibroblast-derived Smo controls kidney fate after AKI. From a novel perspective of cell matrix-cell interactions, our study provides open-access data resources as a complementary piece of the puzzle for understanding AKI pathogenesis.

The detailed information of methodology, usage of chemical and biological reagents, antibodies, and nucleotide sequences of the primers is presented in the Supplementary Methods.

Smo ablation in fibroblasts mitigates ischemic AKI

To elucidate the role of fibroblast-derived Smo in AKI, we generated fibroblast-specific Smo knockout mice by employing the tamoxifen-inducible Cre-LoxP system [9]. Briefly, homozygous Smo-floxed mice were mated with the Gli1-CreER^T2 transgenic mice under the control of endogenous Gli1 promoter to create Smo fibroblast conditional knockout mice (genotype: Cre^+/−, Smo^fl/fl; designated as Gli1-Smo-/-); Gli1 expression is confined to a subset of fibroblasts (Fig. 1A) [3, 9, 25–27]. Age and sex-matched Smo-floxed mice (genotype: Cre^−/−, Smo^fl/fl; designated as Gli1-Smo+/+) from the same litters were used as controls. Gli1-Smo-/- mice were phenotypically normal. There was no appreciable abnormality in kidney function between Gli1-Smo-/- and Gli1-Smo+/+ mice (Supplementary Fig. 1A). After tamoxifen injections for five consecutive days, Cre-mediated recombination was induced in Gli1 + fibroblasts, as previously reported [9, 28, 29]. Mice were then subjected to bilateral renal ischemia-reperfusion injury (IRI) for 1 day to induce AKI after 1-week tamoxifen wash-out (Fig. 1A). Surprisingly, opposite to the results of loss of Shh in tubules,[4] deletion of Smo in Gli1 + fibroblasts preserved kidney function at 1 day after ischemic AKI. Compared to Gli1-Smo+/+ mice, serum creatinine levels were reduced by 22.7% in Gli1-Smo-/- mice (Fig. 1B). Of note, similar result was obtained in a separated ischemic AKI model for 3 days (data not shown). Consistently, Gli1-Smo-/- mice exhibited fewer AKI-associated morphologic changes, such as less cellular debris, brush border loss, and intratubular proteinaceous casts (Fig. 1C; Quantitative data shown in Fig. 1D). Furthermore, the expression of two classic acute tubular injury markers, kidney injury molecule 1 (Kim-1) and neutrophil gelatinase-associated lipocalin (NGAL) [30, 31], were also decreased in Gli1-Smo-/- mice at 1 day after ischemic AKI, compared to Gli1-Smo+/+ mice (Fig. 1E and 1F).

To confirm the above phenotypic changes, we assessed the differences in the key biological mechanisms involved in AKI development, inflammation and cell death [32, 33], between Gli1-Smo+/+ and Gli1-Smo-/- mice ischemic kidneys. Western blots revealed that tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) protein levels were significantly reduced in Gli1-Smo-/- mice, compared with Gli1-Smo+/+ mice (Fig. 1G and 1H). Accordingly, immunostaining results indicated fewer infiltrated CD45⁺ leukocytes, CD3⁺ T cells, and F4/80⁺ macrophages in Gli1-Smo-/- mice diseased kidneys (Fig. 1I, 1J, quantitative data in Fig. 1K, and Supplementary Fig. 1B).

Because AKI features sublethal or lethal damage of renal tubules [33], we further examined if loss of Smo in fibroblasts influences different types of tubular cell death in ischemic kidneys, such as apoptosis, necrosis, or ferroptosis [18], Western blots revealed that pro-apoptosis proteins FasL, Bad, and cleaved caspase-7 were reduced and anti-apoptosis protein Bcl-2 was increased in Gli1-Smo-/- mice, compared with Gli1-Smo+/+ mice [19] (Fig. 1L and 1M). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) showed similar results (Fig. 1N and 1O). Intriguingly, neither ferroptosis marker Glutathione Peroxidase 4 (GPX4) nor necroptosis marker phosphorylated mixed lineage kinase domain-like protein (p-MLKL) was changed between Gli1-Smo+/+ and Gli1-Smo-/- kidneys after ischemic AKI (Supplementary Fig. 1C-1E). Collectively, these results suggested that ablation of Smo in fibroblasts protects against AKI.

Loss of Smo in Gli1 + fibroblasts enhances perivascular mesenchymal cell activity after AKI

Hh/Smo/Gli1 signaling is necessary for fibroblast proliferation in various diseases such as organ fibrogenesis, systemic sclerosis, and cancer microenvironment formation [23, 34–37]. We previously reported that early activated fibroblasts are indispensable for kidney repair after AKI [4]. Therefore, we were puzzled as to why loss of Gli1 + fibroblast-derived Smo alleviated AKI in our current model. Considering Gli1 + fibroblasts are a small fraction of the perivascular mesenchymal cell population [9, 28], we speculated that loss of Smo in Gli1 + fibroblasts may activate the remaining perivascular mesenchymal cells, such as Gli1- fibroblast and pericytes, by stimulating cell survival after AKI. To this end, we systemically assessed the expression of several markers for activated fibroblasts and pericytes in diseased kidneys, including fibroblast-specific protein 1 (FSP1), vimentin, α-smooth muscle actin (α-SMA), and platelet-derived growth factor receptor β (PDGFR-β). Quantitative real-time PCR (qRT-PCR) analyses revealed that mRNA expression of FSP1, vimentin, and α-SMA were significantly increased in Gli1-Smo-/- mice 1 day after ischemic AKI, compared with Gli1-Smo+/+ mice (Fig. 2A). Western blots further demonstrated marked elevation of PDGFR-β, vimentin, and α-SMA proteins in Gli1-Smo-/- mice (Fig. 2B; quantitative data are presented in Supplementary Fig. 2A). Consistently, immunostaining revealed increased expression of FSP-1, PDGFR-β, and vimentin in Gli1-Smo-/- mice diseased kidneys, especially in the juxtamedullary zone (Fig. 2C and Supplementary Fig. 2B-2D). To understand the distribution of proliferative cells in diseased kidneys after loss of Smo in Gli1 + fibroblasts, we assessed proliferating cell nuclear antigen (PCNA) and Ki67 levels. Western blots demonstrated an upregulation of PCNA in Gli1-Smo-/- mice at 1 day after ischemic AKI, compared with Gli1-Smo+/+ mice (Fig. 2B and Supplementary Fig. 2A). Immunohistochemical staining indicated that Ki67 + cells were mainly located in the interstitial compartment of Gli1-Smo-/- kidneys (Fig. 2D-2E). Together, these results indicated that ablation of Smo in fibroblasts in turn activated perivascular mesenchymal cells after ischemic AKI.

Global proteomics identifies nidogen-1 as a key participant in AKI repair after loss of Smo in fibroblasts

To better understand how loss of Smo in fibroblasts mitigates AKI, we used a label-free quantitative approach to profile the global proteome of Gli1-Smo+/+ and Gli1-Smo-/- mice kidneys at 1 day after IRI (Fig. 3A). Impressively, the principal component analysis (PCA) of our proteomic data clearly classified Gli1-Smo+/+ and Gli1-Smo-/- mice according to their genotype (Fig. 3B). A multiple-variation test by ANOVA identified 680 proteins with significantly different expression (Permutation FDR 0.05) between Gli1-Smo+/+ and Gli1-Smo-/- mice diseased kidneys (Quantified proteins are presented in Supplementary Table 1). Compared to Gli1-Smo+/+ mice, 206 and 346 proteins were up- and down-regulated respectively in Gli1-Smo-/- mice diseased kidneys after ischemic AKI (Fig. 3C and Supplementary Table 2; Correlations between biological replicates within the same group and the distribution of protein intensity are presented in Supplementary Fig. 3A-3B). The following Gene Ontology (GO) cellular compartment analysis revealed that these proteins were generally distributed in mitochondrion, extracellular exosome, peroxisome, cytosol, and ECM (Fig. 3D). The activation of perivascular mesenchymal cells in Gli1-Smo-/- mice is redolent of enhanced ECM synthesis in diseased kidneys (Fig. 2). Indeed, unlike most proteins that showed down-regulated trends in other cellular compartments, 110 out of 141 ECM proteins were upregulated in Gli1-Smo-/- mice compared with Gli1-Smo+/+ mice (Fig. 3D). This phenomenon indicates that ECM remodeling by enhanced protein synthesis may play a critical role in Gli1-Smo-/- kidney repair.

We therefore focused on the activated ECM proteins with significant up-regulations in Gli1-Smo-/- mice kidneys. To select the most significantly impacted ECM protein for further investigation, we excluded those that have been well studied [such as vimentin (Fig. 2A-2C) and prelamin-A/C]. Among the remaining proteins, we paid our attention to nidogen-1 (NID1), one of the most prominent matrix proteins upregulated in Gli1-Smo-/- mice (Fig. 3E-3F and Supplementary Fig. 3C) which has been reported as an essential component of the basement membrane that plays a role in cell interactions with the ECM. Since ECM is identified as a major cellular compartment of proteins distributed, we hypothesized that ECM could organize a favorable kidney local microenvironment to repair kidneys after AKI. Thus, we chose NID1 for further study.

To confirm our findings, we assessed NID1 levels in Gli1-Smo+/+ and Gli1-Smo-/- mice diseased kidneys. qPCR analysis indicated that NID1 mRNA level has an upregulation trend (albeit statistically insignificant) in Gli1-Smo-/- mice as compare to Gli1-Smo+/+ (Fig. 3G), and western blots demonstrated a marked elevation of NID1 protein in Gli1-Smo-/- mice (Fig. 3H and Supplementary Fig. 3D), compared with Gli1-Smo+/+ mice. In a separate analysis using single nucleus RNA sequencing, NID1 was highly expressed in fibroblasts and pericytes after IRI at 12 hours (Fig. 3I) [38]. Consistently, immunohistochemical staining confirmed enhanced NID1 distribution in the interstitium of Gli1-Smo-/- ischemic kidneys (Fig. 3J). To further establish the molecular link between NID1 and Smo, we identified multiple potential binding sites of NID1 and Smo in the optimal conformation at pose 51 through virtual protein-protein ZRDOCK and RDOCK algorithms (Supplementary Fig. 4). Immunoprecipitation of Smo or NID1 pulled down NID1 or Smo, respectively, which indicating Smo physically binds to NID1 (Fig. 3K). In addition, to obtain a broader view of the dysregulated pathways after loss of Smo in fibroblasts, we constructed a signaling network based on the STRING database. The analysis with the highest confidence score (cutoff = 0.9) resulted in 413 nodes and 1815 interactions. (Supplementary Fig. 3E). The majority of interactions and nodes were associated with Wnt, Hh, and PPAR-γ signaling pathways and lipid metabolism. These data suggested that loss of Smo in Gli1 + fibroblasts liberated NID1, allowing it to orchestrate a favorable matrix microenvironment after AKI.

Phosphoproteomics reveals that Smo deletion in fibroblasts remodels the Wnt signaling pathway

To gain more insights into the temporal regulation and functional changes in signaling of Smo-deficient fibroblasts in AKI repair, we performed phosphoproteomics on the same set of kidney samples used for the global proteomics (Fig. 4A). Similar to the global proteomics, PCA of the phosphoproteome clearly separated Gli1-Smo+/+ and Gli1-Smo-/- mice kidneys (Fig. 4B). Impressively, most of the significantly different phosphopeptides were decreased in Gli1-Smo-/- mice compared with Gli1-Smo+/+ kidneys (Supplementary Table 3). We then filtered the phosphorylation data set to include those phosphopeptides that were quantified in at least three replicates and used t-test analysis with p-value correnction (Permutation FDR 0.05). Enrichment analysis indicated that phosphoproteins associated with RNA splicing, cell-cell adhesion, and mRNA processing were significantly over-represented (p < 10^− 5). Consistent with network analysis performed with global proteomics (Supplementary Fig. 3E), a clear reduced phosphorylating of multiple components of the Wnt signaling pathway in Gli1-Smo-/- mice was observed (Supplementary Fig. 5A). Pairwise comparisons between Gli1-Smo+/+ and Gli1-Smo-/- mice at 1 day after IRI highlighted significantly changed Wnt components, such as pGSK3β, pSlc9a3r1, pCtnnd1, pTGFβ1i1, pCcny, and pLeo1 (Fig. 4C and Supplementary Fig. 5B-5C). In particular, we identified two downregulated tyrosine phosphorylation sites (Tyr216 and Tyr279) on GSK-3β in Gli1-Smo-/- mice. Dephosphorylation on Tyr216 and Tyr279 represses the activity of GSK-3β. Therefore, in Gli1-Smo-/- mice, inactivated GSK-3β loses its capacity to destabilize β-catenin, a principal protein in the Wnt signaling pathway. This ultimately causes the activation of the canonical Wnt/β-catenin signaling pathway. In addition, the network analysis showed that GSK-3β interacts with other proteins in Gli1-Smo-/- kidneys (Supplementary Fig. 5B).

Previously, we and others reported that activation of the Wnt signaling pathway protected against AKI [8, 10, 12, 39]. Based on the information provided by the phosphoproteome analysis, we assessed the status of the canonical Wnt signaling pathway in Gli1-Smo+/+ and Gli1-Smo-/- mice after AKI. qRT-PCR revealed that 10 out of 19 Wnt family members (Wnt 1, 2, 4, 5A, 5B, 7A, 7B, 9B, 10A, and 11) were significantly induced in Gli1-Smo-/- mice (Fig. 4D). In the same tissue, several Frizzled receptors in the Wnt signaling pathway were also upregulated in Gli1-Smo-/- mice (Supplementary Fig. 6A). Western blots demonstrated that β-catenin, Wnt 1, and Wnt 5A/B proteins in the Wnt pathway were markedly induced in Gli1-Smo-/- mice (Fig. 4E; quantitative data are presented in Supplementary Fig. 6B). Immunohistochemical staining confirmed increased expression of β-catenin, Wnt1, Wnt4, and Wnt5A/B in Gli1-Smo-/- mice diseased tubular cells, compared with Gli1-Smo+/+ mice (Fig. 4F). Of particular interest, three Hh ligands were also induced in Gli1-Smo-/- mice after AKI (Supplementary Fig. 7), although the mechanisms involved remain unclear. Taken together, our data suggest that ablation of Smo in fibroblasts is linked to Wnt signaling pathway activation after ischemic AKI.

Pharmaceutical inhibition of Smo in fibroblasts promotes tubular cell survival through NID1

To further decipher the role of fibroblast-derived Smo in regulating tubular cell survival, we treated normal rat kidney fibroblasts (NRK-49F) with cyclopamine (CPN), a small-molecule, Smo-specific inhibitor. Under hypoxia stress induced by CoCl₂, blocking Smo activity significantly promoted the expression of PDGFR-β, vimentin, α-SMA, and PCNA proteins in cultured fibroblasts (Fig. 5A). NID1 protein was also induced by CoCl₂ and CPN in fibroblasts (Fig. 5B), and immunofluorescence staining of whole cells showed similar results (Fig. 5C).

To validate our in vivo findings that loss of fibroblast-selective Smo induced tubular Wnt signals and reduced cell apoptosis, we performed several in vitro and ex vivo experiments. For our in vitro experiment, we treated normal rat kidney epithelial cells (NRK-52E) with NID1-enriched conditioned medium (CM) collected from fibroblasts (Fig. 5D). Under basal conditions, NID1-enriched CM did not cause dramatic changes in the expression of β-catenin, Wnt1, Wnt2, and Wnt5A/B in NRK-52E cells (Fig. 5E). However, these proteins were significantly induced under hypoxic stress after incubated with NID1-enriched CM (Fig. 5F). Besides NID1-enriched CM, we further applied NID1 recombinant protein to treat NRK-52E at different dosages, as illustrated in Fig. 5D. In this experiment, NID1 activated β-catenin, Wnt1, Wnt5A/B, and Wnt16 in both basal and hypoxic conditions (Fig. 5G and 5H). Impressively, either NID1-enriched CM or NID1 recombinant protein possessed the capacity to reduce tubular cell apoptosis. Specifically, less caspase 3 cleavage was observed in NRK-52E cells after treatment with NID1-enriched CM or NID1 recombinant protein and stimulation with a classic apoptosis inducer, staurosporine (Fig. 5I and 5J). Immunofluorescence staining for cleaved caspase-3 confirmed these findings (Fig. 5K-5N).

To better mimic the in vivo microenvironment, we then isolated decellularized fibroblast scaffolds after hypoxia stress for 24 hours and seeded NRK-52E cells on the scaffold-coated plate (Fig. 6A; as we used in a previous study [40]). After treatment with CPN, NID1 was significantly enriched in the decellularized fibroblast matrix scaffold (Fig. 6B). Similarly, under basal conditions, there were minor differences of β-catenin, Wnt 2, and Wnt 5A/B expression in the seeded tubular cells between control scaffolds and NID1-enriched scaffolds (Fig. 6C). Under hypoxic stress, the NID1-enriched scaffold exhibited enhanced capacity to activate β-catenin, Wnt 2, and Wnt 5A/B and repress caspase-3 cleavage in NRK-52E cells (Fig. 6D-6E). Our results suggest that inhibiting Smo in fibroblasts liberated NID1 to activate tubular Wnt signaling to protect against AKI (Fig. 6F).

Although the kidney is a vulnerable organ that has limited regenerative capacity, there is consensus that the tubular epithelium can repair damage to the kidney after AKI [2, 41]. The mechanisms involved are rigorously debated. For a long time, proximal tubule progenitors were thought to exist in various locations such as bone marrow, the renal interstitium, and the renal papilla [2]. In recent years, the concept of kidney perivascular mesenchymal cells contributing to tubule repair in AKI has been increasingly appreciated [3–5, 9], although many unknowns remain. In this study, combining conditional knockout mice, in vitro and ex vivo models, and systems biology approaches, we illustrated that fibroblasts play an important role in protecting against AKI. Our results indicate that ablation of Smo in fibroblasts 1) mitigates ischemic AKI, 2) promotes perivascular mesenchymal cell activation, 3) upregulates NID1 and activates the Wnt signaling pathway, and 4) NID1 activates Wnt signaling and represses tubular cell apoptosis in vitro and ex vivo.

Smo is one of the multi-span transmembrane proteins in the core reception system for the Hh pathway [42, 43]. It has been well documented that Hh controls cell growth and differentiation through a highly conserved signal transduction pathway which reciprocally travels between producing and responsive cells [18, 25, 44]. In the kidney, deleting Shh in producing tubule cells aggravates AKI [4], whereas the current study shows that loss of Smo in (Hh) responding fibroblasts mitigates tubular cell apoptosis and inflammatory cell infiltration after AKI (Fig. 1). These opposing observations reflect the challenge of elucidating AKI if focused on a single signaling pathway or cell type. What we observed was of particular interest: after ischemic AKI, loss of Smo in Gli1 + fibroblasts activated substantial perivascular mesenchymal cells such as Gli1- fibroblasts, pericytes, and mesenchymal stromal cells (Fig. 2). The number of proliferative interstitial cells is apparently more than tubular cells that exhibited the potential of interstitial cells to retard AKI. But why did the loss of Smo in fibroblasts then activate perivascular mesenchymal cells in vivo after AKI? There are two possibilities. One is that, because our conditional knockout mouse model was induced by an inducible Gli1-Cre-LoxP system, Gli1 + fibroblasts are a small fraction of the entire population of mesenchymal cells [9, 45]. Therefore, the rest of the perivascular mesenchymal cells respond to ischemic stress as usual. Another possibility is, as a group of stem cell-like cells [45], loss of Smo in Gli1 + fibroblasts enhanced cell proliferation and their interaction with neighboring cells after AKI. Of course, the ability of Gli1 + fibroblasts to control the entire process of AKI repair may not be plausible, as the number of Gli1 + fibroblasts in an AKI kidney is limited.

AKI is a refractory clinical syndrome. Therefore, its repair process is certain to involve multi-cellular/non-cellular components. Consistent with the enhanced activation of perivascular mesenchymal cells in Gli1-Smo-/- kidneys (Fig. 2), global proteomics confirmed that ECM is a key cellular compartment where differentially expressed proteins are distributed after ischemic AKI (Fig. 3). As a three-dimensional non-cellular component in the kidney, ECM provides the biophysical scaffolding for the kidney and biochemical or mechanical microenvironment to control cell homeostasis and differentiation [46–48]. The number of significantly upregulated ECM proteins in Gli1-Smo-/- mice is nearly 3.5-fold more than in their littermate controls (Fig. 3). These data suggest perivascular mesenchymal cells synthesized an appropriate amount of ECM to reconstruct kidneys under acute ischemic stress. Among these identified ECM markers, NID1 is one of the most prominent matrix proteins expressed in Gli1-Smo-/- mice. (Fig. 3). NID1, formerly known as entactin, is an essential structural component of the basement membrane and ECM. The basement membrane is a specialized layer of ECM components in the kidney that plays a central role in maintaining organ functions [49]. Our data showed that NID1 is predominantly localized in the interstitium after AKI (Fig. 3). Thus far, most studies have focused on secretion of NID1 preventing cancer metastasis. Sporadic studies have reported that NID1 mitigates ischemia and promotes heart survival and regeneration after injury [50, 51]. Consistent with the positive roles NID1 plays in treating various diseases, our data provides new evidence for a role of NID1 in AKI and kidney repair as well.

Another novel finding of our study is that Smo physically binds NID1 to form a protein-protein complex. As a frizzled-class G-protein-coupled receptor (GPCR), Smo contains two distinct ligand-binding sites: one in its heptahelical transmembrane domain (TMD) and another one in the extracellular cysteine-rich domain [52]. The TMD binding site is known to bind the small-molecule Smo-specific inhibitor, CPN [53]. In comparison, whether a GPCR could physically link to macromolecules like NID1, integrate into the basement membrane, and subsequently give them unique functions in diseased kidneys is unclear. We showed through immunoprecipitation that Smo binds to NID1 (Fig. 3). Therefore, in the absence of Smo, NID1 protein would be released, and then deposited in the neighboring epithelial basement membranes. Basement membranes are essential ECM structures shaped by the networks of collagen type IV and laminins, both of which are linked by NID1 [51]. This strategic subcellular localization of NID1 enables it to mediate cell attachment and communications between cells and ECM [54–56]. We also demonstrated in our in vitro and ex vivo systems, that increased NID1 led to reduced tubular cell apoptosis (Figs. 5 & 6).

Considering the visual pathological effects of AKI repair, it is undeniable that the fibroblast-ECM-tubule interaction is a standard process of building a favorable kidney local microenvironment (KLM), which is complex, heterotypic, and dynamic [57]. We need to ask whether NID1-enriched KLM plays a role in repairing AKI. In this study, we utilized a decellularized scaffold that retains fibroblast's overall architecture to closely imitate the in vivo matrix microenvironment. NID1-enriched scaffold activated the Wnt pathway and attenuated staurosporine-induced tubular cell apoptosis (Fig. 6). Our global and phosphoproteomics data coincidentally provided us strong information that the Wnt signaling pathway was dramatically activated in Gli1-Smo-/- mice after AKI. As we know, at the early phase of AKI, the majority of Wnt family proteins are activated in tubules and exhibit a protective role in preserving renal function after AKI [10, 58]. Therefore, activation of the Wnt pathway in Gli1-Smo-/- mice may be associated with NID1 redistribution to the diseased tubules, to a certain extent; although this complicated mechanism remains ambiguous. In addition, our study also observed the upregulation of all three Hh ligands in Gli1-Smo-/- mice. Further exploration is needed to clarify whether such upregulation is a cause or a consequence of Smo loss in Gli1 + fibroblasts and Wnt activation.

Our study has some limitations. First, the core reception system for Hh signaling includes two multispan transmembrane proteins—Ptc and Smo—and Hh reciprocally controls them. Ptc inhibits Smo in the absence of Hh. In the presence of Hh, Hh binds Ptc to release its inhibition of Smo, leading to Smo phosphorylation and activation. Our study has not evaluated whether loss of fibroblast-derived Smo influences Ptc in AKI repair. Second, our study did not explore the mechanisms of how NID1 participates in GSK-3β tyrosine phosphorylation. Third, our study looked at one time point and the proteomics was performed on whole AKI kidneys. Fourth, the Gli1 promoter in the Cre-LoxP system limited the population of the target fibroblast. In the current study, we cautiously described the kidney Gli1 + cells in relatively broad terms by encompassing perivascular MSCs, pericytes, and fibroblasts. More robust genetic animal models are needed to fully elucidate the roles of perivascular mesenchymal cells in AKI.

In summary, using a multidisciplinary approach, we report that loss of Smo in fibroblasts protects against AKI through ECM-cell interactions. We highlight the importance of creating a favorable tissue microenvironment in determining AKI prognosis. Our findings provide preclinical stage strategies to understand AKI better and shed light on effectively restoring kidney functions.

Data availability

Raw Mass Spectrometry data were deposited in MassIVE with the data set identifier MSV000088609. (doi:10.25345/C5NS23).

Disclosure

The authors declare no conflict of interest.

Acknowledgments

This work was supported by the National Institutes of Health (NIH) grants DK064005, DK106049 (to Liu Y.), DK116816, DK128529 (to Zhou D.). We are grateful to Dr. Benjamin Humphreys lab at Washington University in St. Louis for using their online database. We are grateful to Dr. Justin Radolf at the University of Connecticut, School of Medicine and Dr. Karen E. Nelson at J Craig Venter Institute for reviewing the manuscript.

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Fibroblast-selective smoothened governs the prognosis of acute kidney injury

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